Method for measuring thyroglobulin

ABSTRACT

A method for measuring thyroglobulin(s), comprising using each one or more kinds of proteins capable of binding to a constant region of thyroglobulin(s) and proteins capable of specifically binding to a specific sugar chain structure of thyroglobulin(s) having the specific sugar chain structure, and the method of determining a malignancy of thyroid tumor, a reagent thereof using the obtained by the method for measuring thyroglobulin(s).

BACKGROUND OF THE INVENTION

[0001] The present invention relates to a method for measuringthyroglobulin (hereinafter abbreviated as Tg), more particularly amethod for measuring Tg(s) having a specific sugar chain structure, andfurther relates to a method for determining malignancy of thyroidcarcinoma based upon an amount of Tg(s) having a specific sugar chainstructure.

[0002] It has been known that Tg(s) is a sugar protein of molecularweight of 660,000 composed of a dimer of subunits having molecularweight of 330,000, which is secreted from a thyroid gland and that itscontent in cells or blood is increased in benign or malignant thyroidgland diseases. For this reason, the measurement of Tg(s) in blood hasbeen used in progress observation after an operation on thyroid tumor.However, it is not possible to determine whether the thyroid tumor isbenign or malignant on the basis of the Tg(s) content.

[0003] Thus, determination of malignancy of thyroid tumor has beenconducted by sonography, punch aspiration biopsy, etc. However, therehave been such problems that in the sonography, highly skilled diagnosistechnique is required and in the punch aspiration biopsy, several daysare required in the determination when culture the collected cells, andon the other hand highly skilled technique is required when thedetermination is conducted by observation of cells collected. Further,in the punch aspiration biopsy, there is also such a problem thatdifferentiation of follicular adenoma from follicular carcinoma isdifficult.

[0004] Further, it has been tried that benign or malignant of thyroidtumor is determined by specifying a sugar chain on the surface of thecell with the use of protein recognizing a sugar such as a lectin, andas the result, it has been found that there is observed a difference insugar chain structures between benign and malignant thyroid carcinoma.In this method, however, each cell collected is reacted with a labeledprotein recognizing a sugar chain and then an amount of the labeledmaterial is confirmed under microscope, and thus this method isaccompanied with such problems that skilled technique is required forthe test and quantitative evaluation of the test result is difficult.

[0005] On the other hand, purified Tg(s) have been obtained from benigncell and malignant carcinoma cell and the structures of their sugarchains have been analyzed to find that the structures of sugar chains inthe cells are different between the benign cell and the malignantcarcinoma cell. However, also in this method, there is such a problemthat a long time is required for the purification of Tg(s) from thecells collected and for the analysis of the structures of the sugarchains.

BRIEF SUMMARY OF THE INVENTION

[0006] Under the situation as mentioned above, the problem to be solvedby the present invention is to provide a method for easily and simplymeasuring various kinds of Tg(s) in samples originated from a livingbody, to provide a method for determining malignancy of thyroid tumor onthe basis of the measured result and to provide a reagent for thispurpose.

[0007] The present invention has been accomplished for the purpose ofsolving those problems as mentioned above, and it relates to as follows;

[0008] (1) A method for measuring thyroglobulin(s), comprising usingeach one or more kinds of proteins capable of binding to a constantregion of thyroglobulin(s) and proteins capable of specifically bindingto a specific sugar chain structure of thyroglobulin(s) having thespecific sugar chain structure.

[0009] (2) A reagent for measuring thyroglobulin(s), which compriseseach one or more kinds of proteins capable of binding to a constantregion of thyroglobulin(s) and proteins capable of specifically bindingto a specific sugar chain structure of thyroglobulin(s) having thespecific sugar chain structure.

[0010] (3) A reagent for determining malignancy of thyroid tumor, whichcomprises each one or more kinds of proteins capable of binding to aconstant region of thyroglobulin(s) and proteins capable of specificallybinding to a specific sugar chain structure of thyroglobulin(s) havingthe specific sugar chain structure.

[0011] (4) A method of determining a malignancy of thyroid tumor, whichcomprises measuring one or more of thyroglobulins having a specificsugar chain structures present in a sample originated from a living bodyand determining the malignancy of thyroid tumor on the basis of theamounts of the thyroglobulins.

[0012] (5) A method of determining a malignancy of thyroid tumor, whichcomprises separating thyroglobulins from one another present in a sampleoriginated from a living body on the basis of the difference between oramong their sugar chain structures, measuring the proportion of one ormore of thyroglobulin(s) having a specific sugar chain structure,related to the total amount of thyroglobulin(s), and determining themalignancy of thyroid tumor on the basis of a result of the measurement.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]FIG. 1 shows a ratio (%) of Tg(s) not bound to Concanavalin Arelated to the total Tg(s) in various kinds of thyroid tissue extractsobtained in Example 1.

[0014]FIG. 2 shows a ratio (%) of Tg(s) not bound to Ricinus communisagglutinin-120 related to the total Tg(s) in various kinds of thyroidtissue extracts obtained in Example 2.

[0015]FIG. 3 shows a ratio (%) of Tg(s) not bound to Concanavalin Arelated to the total Tg(s) in various kinds of thyroid tissue extractsobtained in Example 3.

[0016]FIG. 4 shows a ratio (%) of Tg(s) not bound to Lens culinarisagglutinin related to the total Tg(s) in various kinds of thyroid tissueextracts upon using “anti Tg-86” as an anti Tg antibody, which wasobtained in Example 4.

[0017]FIG. 5 shows a ratio (%) of Tg(s) not bound to Lens culinarisagglutinin related to the total Tg(s) lectin in various kinds of thyroidtissue extracts upon using “anti Tg-78” as an anti Tg antibody, whichwas obtained in Example 4.

[0018]FIG. 6 shows a ratio (%) of Tg(s) not bound to Concanavalin Arelated to the total Tg(s)in various kinds of thyroid tissue extracts

DETAILED DESCRIPTION OF THE INVENTION

[0019] The present inventors have extensively studied to solve the abovementioned problems to reach a finding that a total amount of Tg(s) andan amount of Tg(s) having a specific sugar chain structure or that ofTg(s) having a sugar chain structure other than the specific sugar chainstructure in a living sample can be measured by using each one or morekinds of “proteins capable of specifically binding to Tg(s)” and“proteins capable of specifically binding to a specific sugar chainstructure of Tg(s) having the specific sugar chain structure”, and havecontinued to study on the basis of this finding to reach an additionalfinding that the amount of Tg(s) having a specific sugar chain structureand/or that of Tg(s) having a sugar chain structure other than thespecific sugar chain structure, or a ratio of Tg(s) having a specificsugar chain structure or that of Tg(s) having a sugar chain structureother than the specific sugar chain structure to total amount of Tg(s)in a living sample can advantageously be used for determining malignantof thyroid tumor, and on the basis of those findings, the presentinvention has been accomplished.

[0020] The “proteins capable of binding to a constant region of Tg(s)”of the present invention (hereinafter abbreviated as “a Tg-bindingprotein”) includes anti-Tg antibody capable of binding to a constantregion of Tg(s), a receptor capable of binding to Tg(s), etc. Theprotein may be used alone or in a suitable combination of two or morethereof. The constant region of Tg(s) means a region having a structurewhich is common to all Tg(s) in a living sample. The “Tg-bindingprotein” of the present invention may have a capability of binding alsoto a region other than the constant region.

[0021] The “Tg-binding protein” includes one having a characteristic of“proteins capable of binding to Tg(s) but not capable of binding toTg(s) to which the “proteins capable of specifically binding to aspecific sugar chain structure of Tg(s) having the specific sugar chainstructure” (hereinafter abbreviated as “a protein binding to a specificsugar chain”) is already bound (hereinafter abbreviated as a competitiveTg-binding protein”), one having a characteristic of “a protein capableof binding to all Tg(s) regardless whether a protein binding to aspecific sugar chain structure is already bound thereto or not”(hereinafter abbreviated as “a non-competitive Tg-binding protein”).

[0022] The anti-Tg antibody capable of binding to a constant region ofTg(s) may be any polyclonal antibodies prepared by immunizing an animalsuch as horse, sheep, rabbit, goat, rat and mouse with the substance tobe measured according to a conventional method described in TadashiMatsuhashi et al. “Meneki Jikkengaku Nyumon” 2nd. ed., Gakkai-ShuppanCenter Ltd., 1981; or any monoclonal antibodies produced by hybridomasobtained by fusing cells from a tumor cell line of mouse with mousespleen cells previously immunized with the substance to be measuredaccording to a conventional cell fusion method established by G. Köhlerand C. Milstein [Nature, 256, 495 (1975)].

[0023] The “protein binding to a specific sugar chain structure” of thepresent invention includes an antibody, a lectin, etc. which are capableof specifically binding to a specific sugar chain structure of Tg(s).More definitely, including an antibody reactive with Lewis type sugarchain such as anti-Le^(a) antibody, anti-Le^(b) antibody, anti-Le^(x)antibody, anti-Le^(y) antibody, anti-S-Le^(a) antibody and otherantibodies, a lectin capable of binding to L-fucose such as Lotustetragonolobus agglutinin, a lectin capable of binding to D-galactose orN-acetyl-D-galactosamine such as Arachis hypogoea agglutinin, soybeanagglutinin, Ricinus communis agglutinin and phytohemagglutinin, a lectincapable of binding to D-mannose such as Concanavalin A, Lens culinarisagglutinin and Pisum sativum agglutinin, a lectin capable of binding toN-acetylglucosamine such as wheat germ agglutinin and Datura stramoniumagglutinin, a lectin capable of binding to sialic acid such as Limuluspolyphemus agglutinin, etc., among which those capable of binding toD-galactose or N-acetyl-D-galagtosamie and those capable of binding toD-mannose are preferable. The lectin may be used alone or in a suitablecombination of two or more thereof.

[0024] “The capable of binding to a sugar chain structure” in the aboveclassification of the lectin means that a lectin once bound to anaffinity column immobilized a suitable sugar chain can be eluted by thissugar. For instance, a lectin capable of binding to D-galactose orN-acetyl-D-galactosamine means that a lectin once bound to an affinitycolumn can be eluted by this D-galactose or N-acetyl-D-galactosamine.

[0025] The antibody capable of specifically binding to a specific sugarchain structure of Tg(s) having the specific sugar chain structure mayalso be polyclonal antibodies or monoclonal antibodies prepared by aconventional manner as mentioned above.

[0026] The specific sugar chain structure in the present inventionincludes particularly (1) a sugar chain structure to which the abovementioned lectin can be bound and (2) a sugar chain structure containedin Tg(s) which is produced by carcinoma cell such as thyroid carcinoma.More particularly, the sugar chain structures described in documentssuch as Yamamoto, K., Eur. J. Biochem., vol. 143, 133-144, 1984 and soon can be mentioned.

[0027] The measurement method of the present invention is characterizedin that various kinds of Tg(s) in a sample derived from a living bodysuch as plasma, serum, cerebrospinal fluid, extracted solution ofvarious kinds of living tissues and urine are measured by using “aTg-binding protein” and “a protein binding to a specific sugar chainstructure” in a suitable combination, and the definite object to bemeasured includes a total Tg(s) amount(s), an amount of Tg(s) having aspecific sugar chain structure, an amount of Tg(s) having a sugar chainstructure other than the specific sugar chain structure. Tg(s) isdecomposed in a living body to give various kinds of fragments, andthese fragments to which “a Tg-binding protein” and/or “a proteinbinding to a specific sugar chain structure” can be bound, among them,can also be the object to be measured in the present invention.

[0028] Those objects to be measured can be measured separately orsimultaneously in one shot step.

[0029] The follows are working modes of the present invention.

[0030] (1) A method for measuring Tg(s), which comprises

[0031] {circle over (1)} reacting a sample originated from a living bodywith either one of (a) a protein-(1) capable of binding to a constantregion of Tg(s) or (b) a protein-(2) capable of specifically binding toa specific sugar chain structure of Tg(s) having the specific sugarchain structure to form a conjugate of [the protein-(1)]-[Tg] or [theprotein-(2)]-[Tg],

[0032] {circle over (2)} reacting the resulting conjugate with the otherone of (a) the protein-(1) or (b) the protein-(2), which is labeled by alabeling substance, to form a conjugate of [the protein-(1)]-[Tg]-[thelabeled protein-(2)] or [the protein-(2)]-[Tg]-[the labeledprotein-(1)],

[0033] {circle over (3)} separating the conjugate from the free labeledprotein (2) or the free labeled protein-(1), and

[0034] {circle over (4)} measuring an amount of the labeling substancein the conjugate formed in the step {circle over (2)}, whereby an amountof a Tg(s) having the specific sugar chain structure is obtained.

[0035] (2) A method for measuring Tg(s), which comprises

[0036] {circle over (1)} reacting a sample originated from a living bodywith a labeled protein-(2) capable of specifically binding to a specificsugar chain structure of Tg(s) having the specific sugar chain structureand a protein-(3) capable of binding to all Tg(s) regardless whether thelabeled protein-(2) is already bound thereto or not to form a conjugateof [the labeled protein-(2)]-[Tg]-[the protein-(3)], and

[0037] {circle over (2)} separating the conjugate from the free labeledprotein-(2), and

[0038] {circle over (3)} measuring an amount of the labeling substancein the conjugate formed in the step {circle over (1)}, whereby an amountof a Tg(s) having the specific sugar chain structure is obtained.

[0039] (3) A method for measuring Tg(s), which comprises

[0040] {circle over (1)} reacting a sample originated from a living bodywith a protein-(2) capable of specifically binding to a specific sugarchain structure of Tg(s) having the specific sugar chain structure toform a conjugate of [Tg]-[the protein-(2)],

[0041] {circle over (2)} separating the conjugate from Tg(s) to whichthe protein-(2) is not bound, and

[0042] {circle over (3)} measuring an amount of the Tg(s) having thespecific sugar chain structure in the conjugate.

[0043] (4) A method for measuring Tg(s), which comprises

[0044] {circle over (1)} reacting a sample originated from a living bodywith a protein-(1) capable of binding to a constant region of Tg(s) toform a conjugate of [the protein-(1)]-[Tg],

[0045] {circle over (2)} reacting the conjugate with both of aprotein-(2) capable of specifically binding to a specific sugar chainstructure of Tg(s) having the specific sugar chain structure, and thenreacted with a labeled protein-(4) capable of binding to Tg(s) but notcapable of binding to Tg(s) to which the protein-(2) is already bound,to form conjugate (a) of [the protein-(1)]-[Tg]-[the protein-(2)] and aconjugate (b) [the protein-(1)]-[Tg]-[the labeled protein-(4)],

[0046] {circle over (3)} separating the conjugates from the free labeledprotein-(4), and

[0047] {circle over (4)} measuring an amount of the labeling substancein the conjugate (b) formed in the step {circle over (2)}, whereby anamount of Tg(s) having a sugar chain structure other than the specificsugar chain structure is obtained.

[0048] (5) A method for measuring Tg(s), which comprises

[0049] {circle over (1)} reacting a sample originated from a living bodywith a protein-(2) capable of specifically binding to a specific sugarchain structure of Tg(s) having the specific sugar chain structure, andthen reacted with a protein-(4) capable of binding to Tg(s) but notcapable of binding to Tg(s) to which the protein-(2) is already bound toform a conjugate of (a) [the protein-(2)]-[Tg] and a conjugate of (b)[the protein-(4)]-[Tg],

[0050] {circle over (2)} separating the conjugate (b) from others,

[0051] {circle over (3)} reacting the conjugate (b) with a labeledprotein-(1) capable of binding to a constant region of Tg(s) to form aconjugate (c) of [the protein-(4)]-[Tg]-[the labeled protein(1)],

[0052] {circle over (4)} separating the conjugate (c) from free labeledprotein-(1), and

[0053] {circle over (5)} measuring an amount of the labeling substancein the conjugate (c) formed in the step {circle over (3)}, whereby anamount of Tg(s) having a sugar chain structure other than the specificsugar chain structure.

[0054] (6) A method for measuring Tg(s), which comprises

[0055] {circle over (1)} reacting a sample originated from a living bodywith both a protein-(2) capable of specifically binding to a specificsugar chain structure of Tg(s) having the specific sugar chain structureand a labeled protein-(4) capable of binding to Tg(s) but not capable ofspecifically binding to a Tg(s) to which the protein-(2) is alreadybound to form a conjugate of (a) [the protein-(2)]-[Tg] and a conjugateof (b) [the labeled protein-(4)]-[Tg], and

[0056] {circle over (2)} separating the conjugate (b) from the freelabeled protein-(4), and

[0057] {circle over (3)} measuring an amount of the labeling substancein the conjugate (b) formed in the step {circle over (3)}, whereby anamount of Tg(s) having a sugar chain structure other than the specificsugar chain structure is obtained.

[0058] (7) A method for measuring Tg(s), which comprises

[0059] {circle over (1)} reacting sample originated from a living bodywith both a labeled protein-(1) capable of binding to a constant regionof Tg(a) and a protein-(2) capable of specifically binding to a specificsugar chain structure of Tg(s) having the specific sugar chain structureto form conjugates of (a) [the labeled protein-(1)]-[Tg] and (b) [thelabeled protein-(l)]-[Tg]-[the protein-(2)], and

[0060] {circle over (2)} separating the each conjugates from the freelabeled protein-(1), and

[0061] {circle over (3)} measuring the amounts of the labelingsubstances in each of the conjugate (a) and conjugate (b) formed in thestep {circle over (1)} are measured respectively, whereby amounts ofTg(s) having the specific sugar chain structure, Tg(s) having a sugarchain structure other than the specific sugar chain structure and totalTg(s) are obtained.

[0062] (8) A method for measuring Tg(s), which comprises

[0063] {circle over (1)} reacting a sample originated from a living bodywith a protein-(2) capable of specifically binding to a specific sugarchain structure of Tg(s) having the specific sugar chain structure and alabeled protein-(3) capable of binding to all Tg(s) regardless whetherthe protein-(2) is already bound thereto or not, and a protein-(4)capable of binding to Tg(s) but not capable of binding to Tg(s) to whichthe protein-(2) is already bound to form conjugates of (a) [the labeledprotein-(3)]-[Tg]-[the protein-(4)] and (b) [the labeledprotein-(3)]-[Tg]-[the protein-(2)],

[0064] {circle over (2)} removing the free labeled protein-(3), andseparating the conjugates (a) and (b), and

[0065] {circle over (3)} measuring the amounts of the labelingsubstances in each conjugate (a) and (b) formed in the step {circle over(1)}, whereby amounts of Tg(s) having the specific sugar chainstructure, Tg(s) having a sugar chain structure other than the specificsugar chain structure and total Tg(s) are obtained.

[0066] (9) A method for measuring Tg(s), which comprises

[0067] {circle over (1)} reacting a sample originated from a living bodywith a labeled protein-(1) capable of binding to a constant region ofTg(s) and a protein-(2) capable of specifically binding to a specificsugar chain structure of Tg(s) having the specific sugar chain structureand a protein-(4) capable of binding to Tg(s) but not capable of bindingto Tg(s) to which the protein-(2) is already bound to form conjugates of(a) [the labeled protein-(1)]-[Tg]-[the protein-(4)] and [the labeledprotein-(1)]-[Tg]-[the protein-(2)]

[0068] {circle over (2)} removing the free labeled protein-(1), andseparating the conjugate (a) and (b), and

[0069] {circle over (3)} measuring the amounts of the labelingsubstances in each conjugate (a) and (b) formed in the step {circle over(1)}, whereby amounts of Tg(s) having the specific sugar chainstructure, Tg(s) having a sugar chain structure other than the specificsugar chain structure and total Tg(s) are obtained.

[0070] The following are specific examples of the measurement methods.

[0071] I. Methods for Measuring the Objects Separately

[0072] The objects to be measured, namely, a total Tg(s) amount, anamount of Tg(s) having a specific sugar chain structure and an amount ofTg(s) having a sugar chain structure other than the specific sugar chainstructure are respectively measured by the following.

[0073] I-1. Measurement of a Total Tg(s) Amount

[0074] It can be measured by a per se known measurement method using “aTg-binding protein”.

[0075] I-2. Measurement of an amount of Tg(s) having a specific sugarchain structure

[0076] I-2-1) A Method Using “a Tg-binding Protein” Immobilized On AnInsoluble Carrier

[0077] A “Tg-binding protein” immobilized on an insoluble carrier isreacted with a sample originated from a living body such as plasma,serum, cerebrospinal fluid, extracted solution of various kinds ofliving tissues and urine, whereby the following immobilized conjugate isformed.

[0078] Insoluble Carrier—“A Tg-binding Protein”—Tg

[0079] Then, unnecessary co-existing substances are removed by, forinstance, washing, and the immobilized conjugate is reacted further witha “protein binding to a specific sugar chain structure” bound to alabeling substance (hereinafter abbreviated as “a labeled proteinbinding to a specific sugar chain structure”), whereby the followingimmobilized conjugate is formed.

[0080] Insoluble Carrier—“A Tg-binding Protein”—Tg—“A Labeled ProteinBinding To A Specific Sugar Chain Structure

[0081] Then, the immobilized conjugate product is, washed, for instance,to remove a free “labeled protein binding to a specific sugar chainstructure”, and an amount of the labeling substance in the immobilizedconjugate is measured after a suitable measurement method, and theresulting measured value is applied to a calibration curve showing arelationship between an amount of a labeling substance (measured value)and a concentration of Tg(s) which is previously obtained by measuring astandard solution containing a known amount of Tg(s) having a specificsugar chain structure after a similar method, whereby an amount of Tg(s)having a specific sugar chain structure in the sample can be obtained.

[0082] I-2-2) A Method Using “A Protein Binding To A Specific SugarChain Structure” Immobilized On An Insoluble Carrier

[0083] A sample originated from a living body such as plasma, serum,cerebrospinal fluid, extracted solution of various kinds of livingtissues and urine is reacted with “a protein binding to a specific sugarchain structure” immobilized on an insoluble carrier, whereby thefollowing conjugate is formed.

[0084] Insoluble Carrier—“A Protein Binding To A Specific Sugar ChainStructure”—Tg

[0085] Then, unnecessary co-existing substances are removed, forinstance, by washing, and further “a Tg-binding protein” bound to alabeling substance (hereinafter abbreviated as “a labeled Tg-bindingprotein”) is reacted with the conjugate to give the followingimmobilized conjugate.

[0086] Insoluble Carrier—“A Protein Binding To A Specific Sugar ChainStructure”—Tg—“A Labeled Tg-binding Protein”

[0087] Then, the immobilized conjugate product is washed, for instance,to remove a free “labeled Tg-binding protein” and an amount of thelabeling substance in the immobilized conjugate is measured after asuitable measurement method, and the resulting measured value is appliedto a calibration curve showing a relationship between an amount of alabeling substance (measured value) and a concentration of Tg(s) whichis previously obtained by measuring a standard solution containing aknown amount of Tg(s) having a specific sugar chain structure after asimilar method, whereby an amount of Tg(s) having a specific sugar chainstructure in the sample can be obtained.

[0088] I-2-3) A Method Using “A Labeled Protein Binding To A SpecificSugar Chain Structure” and High-performance Liquid Chromatography(HPLC), etc.

[0089] A sample originated from a living body such as plasma, serum,cerebrospinal fluid, extracted solution of various kinds of livingtissues and urine is reacted with “a labeled protein binding to aspecific sugar chain structure” and “a non-competitive Tg-bindingprotein” to form the following conjugate in the sample.

[0090] “a labeled protein binding to a specific sugar chainstructure”-Tg-“a non-competitive Tg-binding protein”

[0091] Then, the conjugate and a free “labeled protein binding to aspecific sugar chain structure” are separated from each other by HPLCpacked with a suitable packing agent, electrophoresis, etc. and anamount of the labeled substance in the conjugate is measured after asuitable method, and the resulting measured value is applied to acalibration curve showing a relationship between an amount of a labelingsubstance (measured value) and a concentration of Tg(s) which ispreviously obtained by measuring a standard solution containing a knownamount of Tg(s) having a specific sugar chain structure after a similarmethod, whereby an amount of Tg(s) having a specific sugar chainstructure in the sample can be obtained.

[0092] I-3. Measurement Of Tg(s) Having A Sugar Chain Structure OtherThan The Specific Sugar Chain Structure

[0093] I-3-1) A Method Using A Free “Protein Binding To A Specific SugarChain Structure”

[0094] At first, a sample originated from a living body such as plasma,serum, cerebrospinal fluid, extracted solution of various kinds ofliving tissues and urine is reacted with “a protein binding to aspecific sugar chain structure” to form a conjugate of Tg(s) having aspecific sugar chain structure and “a protein binding to a specificsugar chain structure” (hereinafter sometimes abbreviated as a “sugarchain-binding Tg”). Then, a sugar chain-binding Tg and Tg(s) to which aprotein binding to a specific sugar chain structure is not bound, inother words, Tg(s) having a sugar chain structure other than thespecific sugar chain structure (hereinafter abbreviated as a“non-binding Tg”), are removed from the sample by a per se knownseparation method such as centrifugation method, gel filtration method,molecular fraction membrane method and electrophoresis method, etc.,whereby a sample containing only a non-binding Tg is prepared.

[0095] An amount of Tg(s) in thus obtained sample containing only anon-binding Tg is measured by a per se known method using “a Tg-bindingprotein” to give an amount of a non-binding Tg.

[0096] The sample containing only a non-binding Tg may also be preparedby treating the sample with affinity chromatography using “a proteinbinding to a specific sugar chain structure” immobilized on an insolublecarrier.

[0097] I-3-2) A Method Using “A Competitive Tg-binding Protein”

[0098] At first, “a Tg-binding protein” immobilized on an insolublecarrier is reacted with a sample originated from a living body such asplasma, serum, cerebrospinal fluid, extracted solution of various kindsof living tissues and urine to form the following immobilized conjugate.

[0099] Insoluble Carrier—A Tg-binding Protein”—Tg

[0100] Then, unnecessary co-existing substances are removed, forexample, by washing, and the immobilized conjugate is reacted with “aprotein binding to a specific sugar chain structure” and further with “acompetitive Tg-binding protein” to which a labeling substance is bound(hereinafter abbreviated as “a labeled competitive Tg-binding protein”)to give the following immobilized conjugate.

[0101] Insoluble Carrier—“A Tg-binding Protein”—Tg—“A Protein Binding ToA Specific Sugar Chain Structure”

[0102] Insoluble Carrier—“A Tg-binding Protein”—Tg—“A LabeledCompetitive Tg-binding Protein”

[0103] Then, a free “labeled competitive Tg-binding protein” is removedby, for example, washing the immobilized conjugates, and an amount ofthe labeled substance in the immobilized conjugates is measured after asuitable method, and the resulting measured value is applied to acalibration curve showing a relationship between an amount of a labelingsubstance (measured value) and a concentration of Tg(s) which ispreviously obtained by measuring a standard solution containing a knownamount of Tg(s) having a sugar chain structure other than the specificsugar chain structure, namely a non-binding Tg after a similar method,whereby an amount of a non-binding Tg in the sample can be obtained.

[0104] I-3-3) A Method Using “A Competitive Tg-binding Protein”Immobilized On An Insoluble Carrier

[0105] At first, a sample originated from a living body such as plasma,serum, cerebrospinal fluid, extracted solution of various kinds ofliving tissues and urine is reacted with “a protein binding to aspecific sugar chain structure” to form “a sugar chain-binding Tg” inthe sample, and then the sample is reacted with “a competitiveTg-binding protein” immobilized on an insoluble carrier to form thefollowing immobilized conjugate.

[0106] Insoluble Carrier—“A Competitive Tg-binding Protein”—ANon-binding Tg

[0107] Then, unnecessary co-existing substances are removed, forinstance, by washing, and the immobilized conjugate is reacted with “alabeled Tg-binding protein” to form the following immobilized conjugate.

[0108] Insoluble Carrier—“A Competitive Tg-binding Protein”—ANon-binding Protein—“A Labeled Tg-binding Protein

[0109] Then, a free “labeled Tg-binding protein” is removed by, forinstance, washing the immobilized conjugate, and an amount of thelabeling substance in the immobilized conjugate is measured after asuitable method, and the resulting measured value is applied to acalibration curve showing a relationship between an amount of a labelingsubstance (measured value) and a concentration of Tg(s) which ispreviously obtained by measuring a standard solution containing a knownamount of a non-binding Tg after a similar method, whereby an amount ofa non-binding Tg in the sample can be obtained.

[0110] I-3-4) A method using “a labeled competitive Tg-binding protein”and HPLC, etc.

[0111] At first, a sample originated from a living body such as plasma,serum, cerebrospinal fluid, extracted solution of various kinds ofliving tissues and urine is reacted with “a protein binding to aspecific sugar chain structure” to form a sugar chain-binding Tg. Then,the resulting sample is reacted with “a labeled competitive Tg-bindingprotein” to form the following conjugate.

[0112] a non-binding Tg—“a labeled competitive Tg-binding protein”

[0113] Then, the conjugate is separated from a free “labeled competitiveTg-binding protein” by HPLC packed with a suitable packing agent,electrophoresis, etc., and an amount of the labeling substance in theconjugate is measured by a suitable method, and the resulting measuredvalue is applied to a calibration curve showing a relationship betweenan amount of a labeling substance (measured value) and a concentrationof Tg(s) which is previously obtained by measuring a standard solutioncontaining a known amount of a non-binding Tg after a similar method,whereby an amount of a non-binding Tg in the sample can be obtained.

[0114] Needless to say, an amount of Tg(s) having a specific sugar chainstructure (sugar chain-binding Tg) can be obtained by subtracting anamount of Tg(s) having a sugar chain structure other than the specificsugar chain structure (a non-binding Tg) from a total Tg(s) amount, andan amount of Tg(s) having a sugar chain structure other than thespecific sugar chain structure (a non-binding Tg) can be obtained bysubtracting an amount of Tg(s) having a specific sugar chain structure(a sugar chain-binding Tg) from a total Tg(s) amount.

[0115] II. A Method For Measuring The Objects Simultaneously In One ShotStep

[0116] II-1. A Method Using “A Labeled Tg-binding Protein” And “AProtein Binding To A Specific Sugar Chain Structure”

[0117] This method can be conducted as follows after a method disclosedin Japanese Patent Publication-Kokai-No. 191027/1995.

[0118] Namely, at first, {circle over (1)} a sample originated from aliving body such as plasma, serum, cerebrospinal fluid, extractedsolution of various kinds of living tissues and urine is reacted with “alabeled Tg-binding protein” and “a protein binding to a specific sugarchain structure”, or {circle over (2)} the sample is reacted with “alabeled Tg-binding protein” and then the resulting reaction solution isfurther reacted with a protein binding to a specific sugar chainstructure” added to the solution, whereby the following conjugates areformed.

[0119] “a labeled Tg-binding protein”—Tg

[0120] “a labeled Tg-binding protein”—Tg—“a protein binding to aspecific sugar chain structure”

[0121] Then, the conjugates and a free “labeled Tg-binding protein” areseparated from each other by using HPLC packed with a suitable packingagent, electrophoresis, etc. and amounts of the labeled substances inthe respective conjugates are measured after a suitable method, and theresulting measured values are applied to a calibration curve showing arelationship between an amount of a labeling substance (measured value)and a concentration of Tg(s) which is previously obtained by measuringstandard solutions containing a known amount of Tg(s) having a specificsugar chain structure and/or Tg(s) having a sugar chain structure otherthan the specific sugar chain structure after a similar method, wherebyan amount of Tg(s) having a specific sugar chain structure, an amount ofTg(s) having a sugar chain structure other than a specific sugar chainstructure and a total amount thereof, namely a total Tg(s) amount, inthe sample can be obtained simultaneously in one step.

[0122] As the method for separation of conjugates and a free “labeledTg-binding protein” from one another, HPLC is preferable because it iseasily handled and can be conducted repeatedly.

[0123] As the “Tg-binding protein”, a non-competitive one is preferable.

[0124] II-2. A Method Using “A Non-competitive Tg-binding Protein”, “ACompetitive Tg-binding Protein” And “A Protein Binding To A SpecificSugar Chain Structure”

[0125] At first, a sample originated from a living body such as plasma,serum, cerebrospinal fluid, extracted solution of various kinds ofliving tissues and urine is reacted with “a non-competitive Tg-bindingprotein” to which a suitable labeling substance is bound (hereinafterabbreviated as “a labeled non-competitive Tg-binding protein”), “acompetitive Tg-binding protein” and “a protein binding to a specificsugar chain structure” to form the following conjugates.

[0126] “a labeled non-competitive Tg-binding protein”—Tg—“a competitiveTg-binding protein”

[0127] “a labeled non-competitive Tg-binding protein”—Tg—“a proteinbinding to a specific sugar chain structure”

[0128] Then, the conjugates and a free “labeled non-competitiveTg-binding protein” are separated from each other by using HPLC packedwith a suitable packing agent, electrophoresis, etc. and amounts of thelabeled substances in the respective conjugates are measured after asuitable method, and the resulting measured values are applied to acalibration curve showing a relationship between an amount of a labelingsubstance (measured value) and a concentration of various kinds of Tg(s)which is previously obtained by measuring standard solutions containinga known amount of Tg(s) having a specific sugar chain structure and/orTg(s) having a sugar chain structure other than the specific sugar chainstructure after a similar method, whereby an amount of Tg(s) having aspecific sugar chain structure, an amount of Tg(s) having a sugar chainstructure other than the specific sugar chain structure and a totalamount thereof, namely a total Tg(s) amount, in the sample can beobtained simultaneously in one step. Further, by allowing “anon-competitive Tg-binding protein” having a different epitope from “thelabeled non-competitive Tg-binding protein” to take a reaction at thesame time, difference between the properties of the conjugates and thoseof serum ingredients can be increased, as a result of which influencesby serum ingredients can be minimized to increase measurement accuracy.In this point of view, this technique is desirable.

[0129] As the method for separation of conjugates and a free “labelednon-competitive Tg-binding protein” from one another, HPLC is preferablebecause it can be conducted repeatedly.

[0130] II-3. A Method Using “A Labeled Tg-binding Protein” And “ACompetitive Tg-binding Protein”

[0131] At first, {circle over (1)} a sample originated from a livingbody such as plasma, serum, cerebrospinal fluid, extracted solution ofvarious kinds of living tissues and urine is reacted with “a labeledTg-binding protein”, and further “a protein binding to a specific sugarchain structure” and “a competitive Tg-binding protein” are reacted withthe above reaction solution to form the following conjugates.

[0132] “a labeled Tg-binding protein”—Tg—“a competitive Tg-bindingprotein”

[0133] “a labeled Tg-binding protein”—Tg—“a protein binding to aspecific sugar chain structure”

[0134] Then, the conjugates and a free “labeled non-competitiveTg-binding protein” are separated from each other by using HPLC packedwith a suitable packing agent, electrophoresis, etc. and amounts of thelabeled substances in the respective conjugates are measured after asuitable method, and the resulting measured values are applied to acalibration curve showing a relationship between the measured values ofa labeling substance and a concentration of various kinds of Tg(s) whichis previously obtained by measuring standard solutions containing aknown amount of Tg(s) having a specific sugar chain structure and/orTg(s) having a sugar chain structure other than the specific sugar chainstructure after a similar method, whereby an amount of Tg(s) having aspecific sugar chain structure, an amount of Tg(s) having a sugar chainstructure other than the specific sugar chain structure and a totalamount thereof, namely a total Tg(s) amount, in the sample can beobtained simultaneously in one shot step. Further, by allowing “aTg-binding protein” having a different epitope from “the labeledTg-binding protein” to take a reaction after the reaction with “alabeled Tg-binding protein”, difference between the properties of theconjugates and those of serum ingredients can be increased, as a resultof which influences by serum ingredients can be minimized to increasemeasurement accuracy. In this point of view, this technique isdesirable.

[0135] As the method for separation of conjugates and a free “labeledTg-binding protein” from one another, HPLC is preferable because it iseasily handled and can be conducted repeatedly.

[0136] The per se known measurement method using “a Tg-binding protein”can be conducted with the use of an anti Tg antibody, for example, asthe “Tg-binding protein” after an immunological assay such as enzymeimmunoassay (EIA), radioimmunoassay (RIA), enzyme-linked immunosorbentassay (ELISA), fluoroimmunoassay (FIA) and a method using HPLC (JapanesePatent Publication-Kokai-No. 301995/1997), and the measurement mechanismmay be any of a sandwich method. a competitive method, a double antibodytechnique method, etc.

[0137] The insoluble carrier used for immobilizing various kinds of “aTg-binding protein” and “a protein binding to a specific sugar chainstructure” may be any of those so far used in the field of the aboveimmunological assay and includes beads, tube, a special tray ormicrotiter plate in which many tubes are molded in a body, etc., whichare made from metal, glass, ceramic, silicone rubber, synthetic polymersuch as polystyrene, polyvinyl chloride, polypropylene, acryl resin andpolymethylmethacrylate, etc., and an immobilizing method may be any ofso far used in the field of immunological assay, such as physicaladsorption methods and chemical binding methods.

[0138] The labeling substance to be bound to “the Tg-binding protein”and “the protein binding to a specific sugar chain structure” is boundincludes alkali phosphatase, β-galactosidase, peroxidase,microperoxidase, glucoseoxidase, glucose-6-phosphate dehydrogenase,acetylcholinesterase, malic dehydrogenase, luciferase and other enzymeswhich are used in EIA, ^(99m)Tc, ¹³¹I, ¹²⁵I, ¹⁴C, ³H and otherradioisotopes which are used in RIA, fluoresceine, dansyl,fluorescamine, coumarin, naphthylamine and their derivatives and otherfluorescent substances which are used in FIA, luciferin, isoluminol,luminol, bis(2,4,6-trifluorophenyl)oxalate and other luminescentsubstances, phenol, naphthol, anthracene and their derivatives and othersubstances which can absorb an ultarviolet light,4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl,3-amino-2,2,5,5-tetramethylpyrrolidine-1-oxyl,2,6-di-t-butyl-α-(3,5-di-t-butyl-4-oxo-2,5-cyclohexadien-1-ylidene)-p-tolyloxyl and other oxyl group-containing compounds which havecharacteristics as spin-labeling agent.

[0139] The method for binding (labeling) the above labeling substance to“the Tg-binding protein” and “the protein binding to a specific sugarchain structure” is bound may be conducted after a per se known one sofar conducted generally in per se known EIA, RIA, FIA, etc. As thelabeling method, a conventional one using a reaction of avidin (orstreptoavidin) with biotin may be used.

[0140] HPLC apparatus used in the measurement method using HPLC in thepresent invention may be any one so far used in this kind of field.

[0141] In the measurement method using HPLC of the present invention,for the purpose of clearer separation of a conjugate from a free“labeled Tg-binding protein” (or “labeled protein binding to a specificsugar chain structure”), use may be made of “the Tg-binding protein” or“the protein binding to a specific sugar chain structure” is bound, towhich a substance enhancing the separation (hereinafter abbreviated as“separation enhancing substance”) disclosed in Japanese PatentPublication-Kokai-No. 191027/1995, No. 301995/1997, etc. is may be used.

[0142] The “separation enhancing substance” used for this purposeincludes preferably proteins such as α-chymotripsinogen,β-galactosidase, lysozyme, cytochrome c and trypsin inhibitor; peptidescontaining an amino acid such as phenylalanin, proline, arginine, ricin,aspartic acid and glutamic acid; halogen atoms such as bromine, chlorineand iodine; synthetic polymers such as polyethylene glycol; polyaminoacids such as polyglutamic acid, polyaspartic acid, polylysin,polyarginine, polyphenylalanin and polytyrosine; alkyl chains having 3to 10 carbon atoms; fatty acids such as palmitic acid, oleic acid andstearic acid; chemical substances having a group reactive with “theTg-binding protein” and “the protein binding to a specific sugar chainstructure” is bound and showing hydrophobic property, such asN-(ε-maleimidocaproyloxy) succinimide (EMCS),N-succinimidyl-6-maleimidohexanoate, Bismaleimidohexane (BMH) andoctylamine, peptides containing a strong acid residue such as4-(p-maleimidophenyl) butylyl Ala-(Tyr(SO₃H))₅ and 4-(p-maleimidophenyl)butylyl Ala-(Tyr(SO₃H))₈ which are disclosed in Japanese PatentPublication-Kokai-No. 301995/1997, etc. The “separation enhancingsubstance” to be used can be selected considering characteristics (suchas pH stability, hydrophobicity, solubility in water and isoelectricpoint) of the objects to be measured such as Tg(s), Tg-binding proteinsand proteins to which a specific sugar chain is bound.

[0143] A method for binding “the separation enhancing substance” with“the Tg-binding proteins” and/or “the proteins binding to a specificsugar chain structure” can be conducted after (1) a per se known mannerfor binding a labeled substance with an antibody generally used in perse known EIA, RIA or FIA (e.g. Yuichi Yamamura “Ikagaku Jikken Koza Vol.8” 1st ed., NAKAYAMA-SHOTEN Ltd., 1971, Akira Kawano “ZusetsuKeikokotai” 1st ed., Soft Science, Inc., 1983; and Eiji Ishikawa,Tadashi Kawai and Kiyoshi Miyai “Koso Men-eki Sokuteiho” 2nd. ed.,IGAKU-SHOIN Ltd., 1982), (2) a per se known manner for modification andbinding of a substance (e.g. Ikuzo Uritani, Kensuke Shimura, MichinoriNakamura and Masaru Funazu “Tanpakushitu-no Kagakushiushoku <Jo>, <Ge>”1st ed., GAKKAO-SHUPPAN CENTER Ltd., 1981; Yuji Inada et al. “Poly(ethylene glycol) Shushoku Tanpakushitsu” Seikagaku vol. 62, No. 11, pp.1351-1362, Japanese Biochemical Association, 1990; and George H K. andMark M. M. “DNA PROBES” STOCKTON PRESS, 1989), etc.

[0144] By a suitable combination of values of a total Tg(s) amount, anamount of Tg(s) having a specific sugar chain structure and Tg(s) havinga sugar chain structure other than the specific sugar chain structure,which are obtained by the measurement method of the present invention,malignancy of thyroid tumor can be determined.

[0145] Namely, for instance, by obtaining a content of Tg(s) having aspecific sugar chain structure or Tg(s) having a sugar chain structureother than the specific sugar chain structure in the total Tg(s),malignancy of thyroid tumor, in other words, as to whether the tumor isbenign or malignant thyroid carcinoma, can be determined.

[0146] Further in detail, by obtaining a content of Tg(s) having aspecific sugar chain structure or Tg(s) having a sugar chain structureother than the specific sugar chain structure in the total Tg(s) withthe use of lectins capable of binding to D-galactose orN-acetyl-D-galactosamine such as Arachis hypogoea agglutinin, soybeanagglutinin, Ricinus communis agglutinin and phytohemagglutinin, lectinscapable of binding to D-mannose such as concanavalin A, Lens culinarisagglutinin and Pisum sativum agglutinin, etc., as the “protein bindingto a specific sugar chain structure” , differentiation of benign thyroidadenoma or Graves' disease, etc. from papillary carcinoma etc., anddifferentiation of follicular adenoma from follicular carcinoma, etc.becomes possible on the basis of the result thus obtained.

[0147] For instance, diagnosis using the extract from a tissue samplecan be performed, taking the following phenomenon into consideration;

[0148] (1) the ratio (%) of Tg(s) not bound to Con A related to theamount of total Tg(s) in sample from the papillary carcinoma issignificantly higher than those in the sample from benign disease suchas benign thyroid adenoma, Grave's disease, follicular adenoma orthyroid adenomatous.

[0149] (2) the ratio (%) of Tg(s) not bound to Con A, related to theamount of total Tg(s) in sample from the follicular carcinoma issignificantly higher than those in the sample from follicular adenomaand normal.

[0150] (3) the ratio (%) of Tg(s) not bound to RCA120 related to theamount of total Tg(s) in sample from the papillary carcinoma issignificantly higher than those in the sample from Grave's disease andnormal.

[0151] (4) the ratio (%) of Tg(s) not bound to LCA related to the amountof total Tg(s) in sample from the papillary carcinoma is significantlyhigher than those in the sample from benign disease such as follicularadenoma, thyroid adnomatous, Grave's disease.

[0152] Namely, the method of determining malignancy of thyroid tumor ofthe present invention has been accomplished on the basis of those factswhich have been found for the first time by the present inventors.

[0153] The reagent for measurement of Tg(s) of the present inventioncomprises each one or more of “proteins capable of binding to a constantregion of Tg(s)” and “proteins capable of specifically binding to aspecific sugar chain structure of Tg(s) having the specific sugar chainstructure”, and the preferable embodiments and specific examples thereofare as mentioned above.

[0154] The reagent for determination of malignancy of thyroid adenomacomprises each one or more of “proteins capable of binding to a constantregion of Tg(s)” and “proteins capable of specifically binding to aspecific sugar chain structure of Tg(s) having the specific sugar chainstructure”, and the preferable embodiments and specific examples thereofare as mentioned above.

[0155] In the reagents, there may be incorporated reagents havinggenerally been used in this field so far as not interfering stability ofco-existing reagents and not interfering the reaction of Tg(s) with“Tg-binding proteins” and/or “proteins binding to a specific sugar chainstructure”, such as a buffering agent, a reaction accelerator and astabilizer such as a sugar, a protein, a salt and a surfactant,antiseptic, etc., and their contents may be selected from a range havingbeen generally used in this field.

[0156] Further, a metal ion such as magnesium has been known as notinfluencing upon activity and stability of lectin, and thus the metalion may also be incorporated in the reagents.

[0157] The buffering agent usable in the reagents of the presentinvention includes all ones having been generally used inimmunoturbidmetry, immunonephelometry, RIA, EIA, etc. such as Trisbuffer, phosphate buffer, veronal buffer, borate buffer and Good'sbuffer, and pH upon measurement is not specifically limited so far as anantibody-antigen reaction, a reaction of Tg(s) with lectin, etc. are notrestrained and generally 6 to 10.

[0158] In the following, the present invention is further explained indetail, and the present invention is not limited thereto by any means.

EXAMPLE 1 A Method for Differentiation of Papillary Tumors UsingConcanavalin A (ConA)

[0159] (Peroxidase Labeled Anti Tg Antibody)

[0160] An anti Tg antibody (hereinafter abbreviated as “anti Tg-1”) wasbound to peroxidase after a conventional manner to give peroxidaselabeled anti Tg antibody (hereinafter abbreviated as “anti Tg-1.POD”).

[0161] (Sample)

[0162] Human thyroid tissue pieces in an amount of 0.1 g (wet wt) werehomogenized in 1 ml of 0.1 M phosphate buffer solution (pH 7.5,containing 0.9% NaCl, hereinafter abbreviated as “PBS”) by ahomogenizer, followed by centrifuging at 4° C. at 30000 g for 5 minutes,and thus obtained supernatant was used as the sample.

[0163] The human thyroid tissues used were 11 papillary carcinomaspecimens, 5 benign thyroid adenoma specimens, 5 Graves' diseasespecimens and 5 normal thyroid tissue specimens.

[0164] (Reagent 1)

[0165] Con A (manufactured and sold by Honen Corp.) was dissolved in PBSto give 15 mg/ml solution, which was used as Reagent 1.

[0166] (Pre-treatment of Sample)

[0167] Each 50 μl of Sample and Reagent 1 were mixed with each other,incubated at 4° C. for overnight and centrifuged at 4° C. at 3000 g for20 min. to remove precipitates, and the resulting supernatant was usedas Pre-treatment solution 1. By the way, Tg(s) bound to Con A wasremoved by the centrifugation.

[0168] PBS was used in place of Reagent 1 after a similar manner to givesupernatant which was used as Pre-treatment solution 2.

[0169] (Measurement of Tg Content)

[0170] PBS in an amount of 1 ml containing 5 μg of an anti Tg antibodyhaving an epitope different from that of “anti Tg-1” (hereinafterabbreviated as “anti Tg-2”) was charged in each of 96 wells of amicroplate, followed by keeping standing at 20° C. for 1 hour andwashing with PBS, whereby “anti Tg-2” was immobilized. Then, 0.2 ml ofPBS containing 1% of fetal bovine serum was added to each of the wells,followed by keeping standing at 20° C. for 1 hour and washing with PBS,whereby blocking treatment was attained. PBS in an amount of 100 μlcontaining 1% of fetal bovine serum and 50 μl of “Pre-treatment solution1” or “Pre-treatment solution 2” were charged to each of the wells,followed by allowing a reaction to take place at 20° C. for 1 hour.After the reaction, each of the wells was washed with PBS, and 100 μl of“anti Tg-1.POD” diluted 10000 times was added thereto, followed byallowing a reaction to take place at 20° C. for 1 hour. After thereaction, the wells were washed with PBS and 100 μl of3,3′,5,5′-tetramethylbenzidine solution (manufactured and sold byKirkegaad and Perry Labs. Inc.) was added thereto, followed by allowinga reaction to take place at 20° C. for 30 minutes and terminating thereaction by addition of 50 μl of 1 M phosphoric acid. Absorbances at 450nm of each of wells were measured by Microplate reader Spectra 1(manufactured and sold by Wako Pure Chemical Industries, Ltd.) and themeasured absorbances were applied to a calibration curve showing arelationship between Tg(s) content and absorbance which was previouslyprepared by using a Tg(s) solution containing a known amount of Tg(s)after a similar manner, whereby Tg(s) contents in “Pre-treating solution1” and “Pre-treating solution 2”.

[0171] Tg(s) content in “Pre-treating solution 1” shows a content ofTg(s) not bound to Con A, and Tg(s) content in “Pre-treating solution 2”shows a total Tg(s) content.

[0172] (Calculation of a Ratio of Tg(s) Not Bound to Con A)

[0173] A ratio (%) of Tg(s) not bound to Con A is calculated by thefollowing equation.

Ratio (%) of Tg(s) not bound to Con A =[(an amount of Tg(s) not bound toCon A)/(total Tg(s))]×100

[0174] (Result)

[0175] Thus obtained ratio (%) of Tg(s) not bound to Con A is shown inFIG. 1.

[0176] As is clear from FIG. 1, a ratio (%) of Tg(s) not bound to Con Ain the extract from tissue of papillary carcinoma is significantlyhigher than those in the extracts from benign thyroid adenoma tissue,Graves' disease tissue and normal thyroid tissue. It is also understoodthat no difference in a ratio (%) of Tg(s) not bound to Con A is foundamong benign thyroid adenoma tissue extract, Graves' disease tissueextract and normal thyroid tissue extract. Namely, it can be recognizedthat an amount of Tg whose sugar chain is changed, in other words, Tg(s)not capable of binding to Con A is increased in papillary carcinomatissue, and thus a ratio (%) of Tg(s) not bound to Con A is very usefulfor differentiation of benign thyroid adenoma from papillary carcinoma.

EXAMPLE 2 A Method for Differentiation of Papillary Carcinoma UsingRicinus communes Agglutinin-120 (RCA120)

[0177] (Peroxidase Labeled Anti Tg Antibody)

[0178] Same as Example 1

[0179] (Sample)

[0180] Sample was prepared by the same manner as in Example 1. The humanthyroid tissues used were 7 papillary carcinoma specimens, 5 Graves'disease specimens and 4 normal thyroid tissue specimens.

[0181] (Reagent 1)

[0182] RCA120 (manufactured and sold by Honen Corp.) was dissolved inPBS to give 2.5 mg/ml solution, which was used as Reagent 1.

[0183] (Pre-treatment of Sample)

[0184] Pre-treatment solution 1 and 2 were prepared by the same manneras in Example 1, except for using Reagent 1 containing RCA 120 insteadof Reagent 1 containing ConA.

[0185] (Measurement of Tg Content)

[0186] Measurement of Tg(s) content was conducted after the same manneras in Example 1.

[0187] Tg(s) content in Pre-treatment solution 1 shows a content ofTg(s) not bound to RCA120 and that in Pre-treatment solution 2 shows atotal Tg(s) content.

[0188] (Calculation Of A Ratio Of Tg(s) Not Bound to RCA120)

[0189] A ratio (%) of Tg(s) not bound to RCA120 is calculated by thefollowing equation.

Ratio (%) of Tg(s) not bound to RCA120=[(an amount of Tg(s) not bound toRCA120/(total Tg(s))]×100

[0190] (Result)

[0191] Thus obtained ratio (%) of Tg(s) not bound to RCA120 is shown inFIG. 2.

[0192] As is clear from FIG. 2, a ratio (%) of Tg(s) not bound to RCA120in the extract of tissue of papillary carcinoma is significantly higherthan those in the extracts of benign goiter tissue, Graves' diseasetissue and normal thyroid tissue. Namely, it can be recognized that anamount of Tg(s) whose sugar chain is changed, in other words, Tg(s) notcapable of binding to RCA120 is increased in papillary carcinoma tissue,and thus a ratio (%) of Tg(s) not bound to RCA120 is very useful fordifferentiation of papillary carcinoma.

EXAMPLE 3 Differentiation of Follicular Carcinoma from FollicularAdenoma Using Con A

[0193] (Peroxidase Labeled Anti Tg Antibody)

[0194] Same as in Example 1

[0195] (Sample)

[0196] Sample was prepared by the same manner as in Example 1. The humanthyroid tissues used were 4 follicular carcinoma specimens, 7 follicularadenoma specimens and 5 normal thyroid tissue specimens.

[0197] (Reagent 1)

[0198] Same as in Example 1

[0199] (Pre-treatment Of Sample)

[0200] Pre-treatment solution 1 and 2 were prepared by the same manneras in Example 1.

[0201] (Measurement Of Tg(s) Content)

[0202] Measurement of Tg(s) content was conducted by the same manner asin Example 1.

[0203] Tg(s) content in Pre-treatment solution 1 shows a content ofTg(s) not bound to Con A and that in Pre-treatment solution 2 shows atotal Tg(s) amount.

[0204] (Calculation Of A Ratio Of Tg(s) Not Bound To Con A)

[0205] A ratio (%) of Tg(s) not bound to Con A was calculated by thesame manner as in Example 1.

[0206] (Result)

[0207] Thus obtained ratio (%) of Tg(s) not bound to Con A is shown inFIG. 3.

[0208] As is clear from FIG. 3, a ratio (%) of Tg(s) not bound to Con Abecomes higher and higher from a normal thyroid tissue extract, then afollicular carcinoma tissue extract, finally to a follicular adenomatissue extract in this order, and it can be understood that there isfound a significant difference in the ratio between a follicularcarcinoma tissue extract and a follicular adenoma tissue extract. Fromthis result, it can be recognized that differentiation betweenfollicular carcinoma and follicular adenoma, which has been difficulteven by cytodiagnosis, can easily be attained by measuring a ratio (%)of Tg(s) not bound to Con A.

EXAMPLE 4 Fractional Measurement of Tg(s) Having Different Sugar ChainStructures Using Lens culinaris Agglutinin (LCA)

[0209] (Peroxidase Labeled Anti Tg Antibody Fab′ Fragment)

[0210] Two kinds of anti Tg antibodies having different recognizableepitopes from each other, which do not bind to Tg bound to LCA(hereinafter abbreviated as “anti Tg-86” and “anti Tg-78”,respectively), were treated after a conventional manner to give Fab′fragments and then they were bound to peroxidase (manufactured and soldby Toyobo Co., Ltd.) after a conventional manner to give peroxidaselabeled anti Tg antibody Fab′ fragments (hereinafter abbreviated as“anti Tg-86.Fab′-POD”, “anti Tg-78.Fab′-POD”, respectively).

[0211] (Antibody Solution 1)

[0212] A 50 mM phosphate buffer solution (pH 7.5, containing 0.15M NaCland 0.5 (w/v) % bovine serum albumin) containing 2 nM of “antiTg-86.Fab′-POD” was prepared, which was used as Antibody solution 1.

[0213] (Antibody Solution 2)

[0214] A 50 mM phosphate buffer solution (pH 7.5, containing 0.15M NaCland 0.5 (w/v) % bovine serum albumin) containing 3 nM of “antiTg-78.Fab′-POD” was prepared, which was used as Antibody solution 2.

[0215] (Reagent 1)

[0216] A 50 mM phosphate buffer solution (pH 7.5, 0.15 M NaCl)containing 15 μM of LCA (manufactured and sold by Honen Corp.) wasprepared, which was used as Reagent 1.

[0217] (Sample)

[0218] Human thyroid tissue pieces in an amount of 0.1 g (wet wt) werehomogenized in 1 ml of 0.1 M phosphate buffer (pH 7.2, containing 0.9%of NaCl) using a homogenizer, followed by centrifuging at 4° C. at100000 g for 60 minutes, and the supernatant obtained was diluted 200 to1100 times with 50 mM phosphate buffer (pH 7.5, containing 0.15 M NaCland 0.5 (w/v) % of bovine serum albumin) to give the sample.

[0219] The human thyroid tissues used were 4 benign disease specimens (1follicular adenoma specimen, 2 adenomotous specimens and 1 Graves'disease) and 4 papillary carcinoma specimens.

[0220] (Measurement Method)

[0221] Sample in an amount of 25 μl was mixed with 15 μl of Reagent 1,followed by allowing a reaction to take place at 8° C. for 1 hour. Thereaction solution in an amount of 15 μl was mixed with 90 μl of Antibodysolution 1, followed by allowing a reaction to take place further at 8°C. for 30 minutes. The resulting reaction solution in an amount of 50 μlwas analyzed using high-performance liquid chromatography (HPLC) underthe conditions as mentioned below to measure an amount of Tg(s) notbound to LCA. The same measurement for the total Tg(s) amount wasconducted on the same sample using 50 mM of phosphate buffer (pH 7.5,containing 0.15M NaCl and 0.5 (w/v) % bovine serum albumin) in place ofReagent 1 containing LCA. Those two results were applied to thefollowing equation to calculate a ratio (%) of Tg(s) not bound to LCAupon using “anti Tg-86.Fab′-POD”.

A ratio (%) of Tg(s) not bound to LCA =[(an amount of Tg(s) not bound toLCA)/(total Tg(s))]×100

[0222] A ratio (%) of Tg(s) not bound to LCA upon using “antiTg-78.Fab′-POD” was also calculated by the same manner except for usingAntibody solution 2 in place of Antibody solution 1.

[0223] (HPLC Conditions)

[0224] Column: Wakopack Wakosil®-5Diol-200 8.0 mm×300 mm (w)

[0225] Eluent: 50 mM phosphate buffer, pH 7.5, 0.15 M NaCl

[0226] Substrate solution: 15 mM citrate buffer (pH 5.5, 313 mMacetoaminophenol (manufactured and sold by Dojin Laboratories,containing 0.12% H₂O₂)

[0227] Flow rate: the eluent 1.0 mL/min, the substrate solution 0.1mL/min

[0228] Detection: Ex 328 nm, Em 432 nm

[0229] (Result)

[0230] Ratios (%) of Tg(s) not bound to LCA upon using “antiTg-86.Fab′-POD” and “anti Tg-78.Fab′-POD” are shown in FIG. 4 and FIG.5, respectively.

[0231] It is understood from FIG. 4 and FIG. 5 that the ratio (%) ofTg(s) not bound to LCA in a papillary carcinoma tissue extract issignificantly higher than that in a benign disease tissue extract.

[0232] Namely, it is understood that differentiation diagnosis ofpapillary carcinoma from benign disease becomes possible by using LCA.

Example 5 Fractional Measurement of Tg(s) Having Different Sugar ChainStructures Using Con A

[0233] (Peroxidase Labeled Anti Tg Antibody Fab′ Fragment)

[0234] “Anti Tg-86.Fab′-POD” prepared in Example 4 was used.

[0235] (Antibody Solution 1)

[0236] A 50 mM phosphate buffer solution (pH 7.5, containing 0.15 M NaCland 0.5 (w/v) % bovine serum albumin) containing 4 nM of “antiTg-86.Fab′-POD” was prepared which was used as Antibody solution 1.

[0237] (Reagent 1)

[0238] A 50 mM phosphate buffer solution pH 7.5, 0.15 M NaCl, containing15 μM of Con A (manufactured and sold by Honen Corp.) was prepared whichwas used as Reagent 1.

[0239] (Sample)

[0240] The same one as in Example 4 was used.

[0241] (Measurement Method)

[0242] Sample in an amount of 10 μl was mixed with 45 μl of Reagent 1,followed by allowing a reaction to take place at 8° C. for 1 hour. Thereaction solution was mixed with 45 μl of Antibody solution 1, followingby allowing a reaction to take place further at 8° C. for 30 minutes.The resulting reaction solution in an amount of 50 μl was analyzed usinghigh-performance liquid chromatography (HPLC) under the conditions asmentioned below to measure an amount of Tg(s) not bound to Con A. Thesame measurement for the total Tg(s) amount was conducted on the samesample using 50 mM of phosphate buffer (pH 7.5, containing 0.15M NaCland 0.5 (w/v) % bovine serum albumin) in place of Reagent 1. Those tworesults were applied to the following equation to calculate a ratio (%)of Tg(s) not bound to Con A.

A ratio (%) of Tg(s) not bound to Con A =[(an amount of Tg(s) not boundto Con A)/(total Tg(s))]×100

[0243] (HPLC Conditions)

[0244] Same as in Example 4

[0245] (Result)

[0246] The obtained ratio (%) of Tg(s) not bound to Con A is shown inFIG. 6.

[0247] It is understood from FIG. 6 that a ratio of Tg(s) not bound toCon A in papillary carcinoma tissue extract is significantly higher thanthat in a benign disease tissue extract.

[0248] Namely, it is understood that differentiation diagnosis ofpapillary carcinoma from benign disease becomes possible by using Con A.

[0249] The present invention is to provide a method for measuring simplyand at high accuracy various kinds of Tg(s) in various kinds livingsamples and a reagent for this method, and differentiation of papillarycarcinoma from benign thyroid adenoma and differentiation of follicularcarcinoma from follicular adenoma can be conducted by using a suitablecombination of the measurement results of various kinds of Tg(s).

What is claimed is:
 1. A method for measuring thyroglobulin(s),comprising using each one or more kinds of proteins capable of bindingto a constant region of thyroglobulin(s) and proteins capable ofspecifically binding to a specific sugar chain structure ofthyroglobulin(s) having the specific sugar chain structure.
 2. Themethod according to claim 1, wherein the thyroglobulin(s) to be measuredare a total thyroglobulin(s), a thyroglobulin having a specific sugarchain structure and/or a thyroglobulin having a sugar chain structureother than the specific sugar chain structure.
 3. The method accordingto claim 1, wherein the specific sugar chain structure is a sugar chainstructure capable of binding to a lectin.
 4. The method according toclaim 3, wherein the lectin is one capable of binding to D-galactose orN-acetyl-D-galactosamine, or binding to D-mannose.
 5. The methodaccording to claim 3, wherein the lectin is Concanavalin A, Lensculinaris agglutinin or Ricinus communis agglutinin.
 6. A reagent formeasuring thyroglobulin(s), which comprises each one or more kinds ofproteins capable of binding to a constant region of thyroglobulin(s) andproteins capable of specifically binding to a specific sugar chainstructure of thyroglobulin(s) having the specific sugar chain structure.7. A reagent for determining malignancy of thyroid tumor, whichcomprises each one or more kinds of proteins capable of binding to aconstant region of thyroglobulin(s) and proteins capable of specificallybinding to a specific sugar chain structure of thyroglobulin(s) havingthe specific sugar chain structure.
 8. A method of determining amalignancy of thyroid tumor, which comprises measuring one or more ofthyroglobulins having a specific sugar chain structures present in asample originated from a living body and determining the malignancy ofthyroid tumor on the basis of the amounts of the thyroglobulins.
 9. Themethod according to claim 8, wherein the malignancy of thyroid tumor isdetermined on the basis of the proportion of one or more ofthyroglobulins having a specific sugar chain structure, related to thetotal amount of thyroglobulin(s).
 10. The method according to claim 8,wherein the measurement of thyroglobulin(s) is done after one or more ofthyroglobulins are separated from one another on the basis of thedifference between or among their sugar chain structure.
 11. The methodaccording to claim 10, wherein the separation is performed by using eachone or more kinds of proteins capable of binding to a constant region ofthyroglobulin(s) and proteins capable of specifically binding to aspecific sugar chain structure of thyroglobulin(s) having the specificsugar chain structure.
 12. The method according to claim 8, wherein thespecific sugar chain structure is a sugar chain structure capable ofbinding to a lectin.
 13. The method according to claim 12, wherein thelectin is one capable of binding to D-galactose orN-acetyl-D-galactosamine, or binding to D-mannose.
 14. The methodaccording to claim 12, wherein the lectin is Concanavalin A, Lensculinaris agglutinin or Ricinus communis agglutinin.
 15. The methodaccording to claim 8, wherein the specific sugar chain structure is onefound in thyroglobulin(s) which is produced by a carcinoma cell.
 16. Themethod as claimed in claim 15, wherein the carcinoma cell is originatedfrom thyroid carcinoma.
 17. A method of determining a malignancy ofthyroid tumor, which comprises separating thyroglobulins from oneanother present in a sample originated from a living body on the basisof the difference between or among their sugar chain structures,measuring the proportion of one or more of thyroglobulin(s) having aspecific sugar chain structure, related to the total amount ofthyroglobulin(s), and determining the malignancy of thyroid tumor on thebasis of a result of the measurement.
 18. The method according to claim17, wherein the separation is performed by using each one or more kindsof proteins capable of binding to a constant region of thyroglobulin(s)and proteins capable of specifically binding to a specific sugar chainstructure of thyroglobulin(s) having a specific sugar chain structure.